Methods for universal target capture

ABSTRACT

The invention generally relates to methods for universal target capture.

RELATED APPLICATION

The present application claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/739,567 filed Dec. 19, 2012,the content of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The invention generally relates to methods for universal target capture.

BACKGROUND

Blood-borne pathogens are a significant healthcare problem. A delayed orimproper diagnosis of bacterial infection can result in sepsis—a seriousand sometimes deadly inflammatory response. Sepsis is among the tenleading causes of death in the United States. See Martin, et al., 2003,NEJM, 348:1546-1554. Exports report that sepsis causes more deaths peryear than prostate cancer, breast cancer and HIV/AIDS combined and thatsepsis is the most important cause of death in intensive care units.

Early detection of bacterial infections is the key to preventing theonset of sepsis. Traditional methods of detecting blood-borne infectionsinclude lab cultures that require days to complete. Other molecularmethods of detecting bacteria require the bacteria to first be capturedand the DNA isolated from the captured bacteria. Capturing the bacteriatypically requires prior knowledge about the bacteria that is sought tobe detected. That information is not always available, particularly whena patient comes to a healthcare facility having an unknown infection.

SUMMARY

The invention provides universal target capture methods. Methods of theinvention involve introducing an agent to a sample that causes anunknown target in the sample to display a universally recognizedelement. Accordingly, any capture moiety that is able to bind to theelement can be used to isolate the target from the sample, regardless ofthe type of target in the sample. Methods of the invention areparticularly useful for isolating unknown microorganisms from a sampleby causing the microorganisms to display a certain protein that isrecognized by an antibody, so that the antibody can be used to capturethe microorganism in the sample.

The invention provides methods and devices for isolating a target from asample by introducing to the sample a binding element that is displayedon the target. The invention is especially useful in samples comprisingmultiple different targets, in which case the binding element isincorporated into all targets and presents a common binding element forisolation of all targets in the sample. In a preferred embodiment, thetarget is a pathogen or a plurality of pathogens. For example, if thepathogen is a bacterium, methods of the invention contemplate theintroduction of a vector (e.g., a virus) to induce the expression of anantigen in the pathogen(s) for binding by, e.g., an antibody in order tofacilitate isolation of the pathogen(s). Viruses capable of acting on arange of target pathogens allows a broad range of unknown pathogens tobe discovered, detected, and/or isolated. A virus, a cocktail ofdifferent viruses, other agents such as plasmids or bacterial ghosts, orcombinations thereof are useful to induce common attachment orexpression of binding elements. The use of a virus or vector thatattaches to a broad range of pathogens, causing display of a commonbinding element is one example of implementation of the invention. Thus,methods are provided that are useful for the early detection ofbacterial infections with high sensitivity, even when pathogen identityis unknown. Methods of the invention allow early detection andcharacterization of an infection (e.g., a bacterial infection), thusreducing the incidence and impact of negative effects of the infection(e.g. sepsis).

In certain aspects, the invention provides methods of isolating pathogenin which a virus or preparation of viruses are introduced to a samplesuspected of containing a pathogen. The virus or viral preparation isused to cause the pathogens to each present a binding element. Pathogensare then separated from the sample using a binding partner that isspecific for the binding element. In one embodiment, the viralpreparation contains one or more vectors or viruses such as abacteriophage including, for example, a broad-host-range bacteriophage,a functionalized bacteriophage such as a biotinylated bacteriophage, aphasmid, a phagemid, a plasmid, or a bacterial ghost.

In one example, a bacteriophage transfects a bacterial pathogen andcause it to express a cell-surface antigen. Where phage transfectioninduces the pathogen to express a cell-surface antigen, thetarget-specific binding partner may include an antibody. In someembodiments, a bacteriophage displays a peptide on its capsid. Where thedisplayed peptide is an antigen, the target-specific binding partner mayinclude an antibody. In certain embodiments, the peptide displayed onthe bacteriophage capsid is biotinylated and the target-specific bindingpartner comprises avidin or streptavidin.

Target-specific binding elements include any member of a binding pairknown in the art and capable of being expressed in a pathogen.Non-limiting examples include antibodies, biotin, avidin, streptavidin,carbohydrates, lectins, hormones, digoxigenin, anti-digoxigenin, andothers. The binding partner may further include a solid substrate suchas a bead, a surface, or a magnetic bead.

The binding element displayed on the target binds to the bindingpartner, for example, through antibody-antigen binding or throughstreptavidin-biotin binding. The binding partner can then be separatedfrom the sample by exploiting the properties of a solid substrate. Wherethe solid substrate includes a fixed surface, such as a wall within amicrofluidic channel or beads in a column, the sample can be washed out.Where the solid substrate includes magnetic beads, magnetic separationtechniques may be used. For example, a magnet can be used to capturetarget/particle complexes and the excess sample washed away.

In other embodiments, a common binding partner is attached to pathogenin a sample via a binding element that is common to the pathogen classto be detected. Attachment chemistries are known in the art and dependupon the nature of the pathogen to be detected.

Methods of the invention are not limited to pathogen detection and canbe used to detect any target in a sample. For example, methods of theinvention are useful for detection of environmental targets in samplesobtained, e.g., from soil, water and other elements in the environment.Common binding partners are introduced as described above or can beintroduced by chemical attachment via a linker or other binding moiety.Methods of the invention are also useful for detection of biohazards insamples obtained in the environment or in samples suspected ofcontaining biohazards introduced by humans (e.g., anthrax and the like).

Methods of the invention may be employed to separate two or more unknownpathogens from a sample when those pathogens are initially present inlow concentrations. In some embodiments, one or both of the pathogens ispresent at a concentration beneath 1 CFU/mL of sample prior toseparation. The separation may be performed using lab techniques such asaffinity purification. In some embodiments, the separation is performedusing a fluidic chip. Once separated, the pathogens may be cultureddetected using a technique such as NMR, PCR, mass spectrometry,impedance measurement, visual detection, or other means.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 gives a diagram of methods for isolating different pathogens.

FIG. 2 illustrates inducing the expression of a common antigen by aviral preparation.

FIG. 3 shows using a viral preparation to biotinylate different unknownpathogens.

FIG. 4 illustrates phage display of a common antigen by differentunknown pathogens.

FIG. 5 illustrates a fluidic cartridge according to certain embodiments.

FIG. 6 gives a perspective view of the cartridge shown in FIG. 5.

FIG. 7 gives a schematic diagram of the cartridge shown of FIG. 5.

DETAILED DESCRIPTION

The invention generally relates to methods and devices for extractingone or more targets from a sample by causing targets in the sample topresent a common binding element. The target can then be isolated usinga binding partner that specifically binds to the binding element; suchas an antibody binding to an antigen or streptavidin binding to biotin,and others known in the art. In one embodiment, the binding element isattached to a substrate, enabling the sample to be washed, leavingbehind the target to be detected. In a preferred embodiment, the targetsare pathogens or environmental hazards.

Methods of the invention can involve using a magnetic particle as asubstrate attached to a binding partner, thereby forming atarget/magnetic particle complex. Methods of the invention can furtherinvolve applying a magnetic field to capture the target/magnetic complexon a surface and wash away the sample. The invention further providesdevices for extracting the pathogens. Devices of the invention caninvolve fluidic channels and chambers having macrofluidic dimension,microfluidic dimensions, or both.

FIG. 1 provides an exemplary diagram of certain methods for isolatingdifferent pathogens. A sample that contains two or more differentpathogens is obtained 101. Methods of the invention may be used toisolate or extract different unknown pathogens from any heterogeneoussample. For example, the sample may include a bodily tissue or fluid. Inparticular embodiments, the sample is a bodily fluid. A bodily fluidrefers to a liquid material derived from, for example, a human or othermammal. Such bodily fluids include, without limit, mucus, blood, plasma,serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid,mammary fluid, urine, sputum, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A bodily fluid may also be a fine needleaspirate. A bodily fluid may also be media containing cells orbiological material.

Methods of the invention are suitable for use with any type of sampleincluding one or more different pathogens including unknown pathogens.The target pathogen refers to the substance in the sample that will becaptured and isolated by methods of the invention. Other targets may becaptured by methods and devices of the invention. The target may bebacteria, fungi, a protein, a cell (such as a cancer cell, a white bloodcell, a virally infected cell, or a circulating fetal cell), a virus, anucleic acid (e.g., DNA or RNA), a receptor, a ligand, a hormone, adrug, a chemical substance, or any molecule known in the art.

In certain aspects, the target pathogen is a bacterium or two or moredifferent bacteria. Methods of devices of the invention can be used toisolate or extract known bacteria, unknown bacteria, or a combinationthereof. Both gram positive bacteria, gram negative bacteria, archaea,or eukaryotes can be isolated using the methods disclosed herein.Specific genera of pathogens that may be sought and assayed for usingthe disclosed methods include Alphaproteobacteria, Bacillus,Betaproteobacteria, Bifidobacterium, Borrelia, Campylobacter, Candida,Citrobacter, Clostridium, Enterobacter, Enterococcus, Escherichia,Flavobacterium, Fusobacterium, Gammaproteobacteria, Klebsiella,Kluyvera, Lactobacillus, Legionella, Leuconostoc, Listeria, Micrococcus,Mycobacterium, Neisseriaceae, Pediococcus, Pneumococcus, Porphyromonas,Prevotella, Propionibacterium, Proteus, Rhodospirillum, Rickettsia,Saccharomyces, Salmonella, Serratia, Shigella, Sphaerotilus,Staphylococcus, Streptococcus, Thermoanaerobacter, Thermoproteus,Vibrio, and Yersinia.

Organisms that can be assayed for with methods and devices of theinvention include Acinetobacter calcoaceticus, Aeromonas hydrophilia,Bacillus anthracis, Bacillus subtilis, Candida albicans, Citrobacterfreundii, E. coli, Enterobacter aerogenes, Enterobacter cloacae,Enterococcus faecium, Fusobacterium nucleatum, Klebsiella oxytoca,Klebsiella pneumoniae, Klebsiella travesanii, Kluyvera ascorbata,Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus fermentum,Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillussalivarius, Listeria monocytogenes, Micrococcus luteus, Pediococcusparvulus, Porphyromonas gingivalis, Prevotella intermedia,Propionibacterium freudenreichii, Proteus vulgaris, Pseudomonasaeruginosa, Rhodospirillum rubrum, Rickettsia conorii, Saccharomycescerevsiae, Salmonella agona, Salmonella enteritidis, Salmonellaheidelberg, Salmonella infantis, Salmonella minnesota, Salmonellamontevideo, Salmonella ohio, Salmonella typhimurium, Serratiamarcescens, Shigella flexneri, Sphaerotilus natans, Staphylococcusaureus, Staphylococcus epidermis, Staphylococcus xylosus, Streptococcusfaecalis, Streptococcus pyrogenes, Thermoproteus tenax, Vibrio cholerae,and Yersinia pseudotuberculosis. Any combination of organisms may be thetarget of methods and devices of the invention and the target of anassay will vary depending on the suspected pathogen or pathogens to beisolated.

In particular embodiments, the sample is a blood sample. Methods of theinvention allow for different unknown pathogens in a blood sample to beisolated and detected at a level as low as or even lower than 1 CFU/ml.Blood may be collected in a container, such as the blood collection tubesold under the trademark VACUTAINER by BD (Franklin Lakes, N.J.). Incertain embodiments, a solution is added that prevents or reducesaggregation of endogenous aggregating factors, such as heparin in thecase of blood. A particular advantage of the methods described herein isthe ability to capture and isolate different unknown pathogens directlyfrom blood samples at low concentrations that are characteristic ofclinical samples (as low as 1 CFU/ml of bacteria in a blood sample).

As shown in FIG. 1, once the sample is obtained 101, a viral preparationis introduced 107 into the sample. The viral preparation includes atleast one virus that attaches 113 to the pathogens. As a result, thepathogens present 119 a binding element.

Any viral preparation may be used that causes different unknownpathogens to present a binding element. In some embodiments, the viralpreparation contains a cocktail of bacteriophage. The cocktail containsdifferent phage viruses that infect different bacteria. In certainembodiments, a broad-host-range phage is used that can infect differentbacteria.

Phages are viruses that can be used as a transduction vector tointroduce genetic material into the different pathogens and induceexpression of, for example, a common antigen. Phage can be used astransduction vectors to introduce exogenous (non-viral) genetic materialinto the pathogens. See U.S. Pat. No. 7,148,054 for discussion. Duringlytic infection with a phage, DNA is packaged into phage heads as phageparticles are formed. Promiscuous high-transducing (HT) mutants of P22which efficiently package DNA with little sequence specificity areknown.

As diagrammed in FIG. 1, infection involves attachment 113 of the phageparticle to a target cell. When transduction is successful, theexogenous DNA is received in the target cell and expressed 119. Forexample, RecA-mediated homologous recombination following injection ofthe donor fragment can result in the inheritance of donor traits.

Any suitable phage or combination of phages can be used in methods ofthe invention. Examples of transducing phage vectors includebacteriophages P1 and P22 of E. coli and S. typhimurium, respectively.Other phages are suitable for use with methods of the invention. Forexample, the bacteriophage Mu can be used to insert DNA into a targetpathogen genome for expression 119. See Groisman, 1991, In vivo geneticengineering with bacteriophage Mu, Methods in Enzymology 202:180-212.Further, hybrid phages such as Mud-P22 may be used. Mud-P22 is a hybridcombining features of phage Mu and P22. Mud-P22 can be inserted atessentially any desired site on the Salmonella chromosome. See Crain,2007, Mud-P22, Methods Enzymology 421:249-59. Other exemplary phages(and their targets) include T4 (E. coli), T5 (E. coli), λ phage (E.coli), T7 phage (E. coli), G4 (E. coli), P1 (E. coli), φ6 (Pseudomonas),Thermoproteus tenax virus 1 (Thermoproteus tenax), M13 (E. coli), MS2(E. coli), Qβ (E. coli), φX174 (E. coli), Φ29 (Bacillus), PZA(Bacillus), Φ15 (Bacillus), BS32 (Bacillus), B103 (Bacillus), M2Y (M2)(Bacillus), Nf (Bacillus), GA-1 (Bacillus), FWLBc1 (Bacillus), FWLBc2(Bacillus), FWLLm3 (Listeria), B4 (Propionibacterium).

Phages are discussed in Chopin, et al., 2002, J Bact 184(7):2030-2033;Muramatsu et al., 1991, Two generalized transducing phages in Vibrioparahaemolyticus and Vibrio alginolyticus, Microbiol Immunol 35(12):1073-1084; Regue et al., 1991, A generalized transducing bacteriophagefor Serratia marcescens, Res Microbiol 42(1):23-27; Kiesel et al., 1993,Phage Acm1-mediated transduction in the facultatively methanol-utilizingAcetobacter methanolicus MB 58/4, J. Gen Virol 74(9):1741-1745; Zhang,et al., 2012, Food Microbiol 31(1):133-36; U.S. Pat. No. 7,732,150; U.S.Pub. 2009/0246752; U.S. Pub. 2009/0047254; and U.S. Pub. 2004/0156831,the contents of each of which are incorporated by reference.

Exemplary phages are further discussed in Welker, 1988, Transduction inBacillus stearothermophilus, J. Bacteriol, 176(11):3354-3359; Darzins etal., 1989, Mini-D3112 bacteriophage transposable elements for geneticanalysis of Pseudomonas aeruginosa, J. Bacteriol 171(7):3909-3916;Blahova et al., 1994, Transduction or imipenem resistance by the phageF-116 from a nosocomial strain of Pseudomonas aeruginosa isolated inSlovakia, Acta Virol 38(5):247-250; Weiss et al., 1994, Isolation andcharacterization of a generalized transducing phage for Xanthomonascampestris pv. campestris, J. Bacteriol 176(11): 3354-3359; Schicklmaieret al., 1995, Frequency of generalized transducing phages in naturalisolates of the Salmonella typhimurium complex, Appl Environ Microbiol61(4): 61(4): 1637-1640; Humphrey et al., 1997, Purification andcharacterization of VSH-1, a generalized transducing bacteriophage ofSerpulina hyodysenteriae, J Bacteriol 179(2):323-329; Willi et a., 1997,Transduction of antibiotic resistance markers among Actinobacillusactinomycetemcomitans strains by temperate bacteriophages Aa phi 23,Cell Mol Life Sci 53(11-12):904-910; Nedelmann et al., 1998, Generalizedtransduction for genetic linkage analysis and transfer of transposoninsertions in different Staphylococcus epidermidis strains,Zentiviralalbl Bakteriol 287(1-2):85-92; Int. Pat. Application Pub. WO2003/035889; U.S. Pat. No. 8,071,337 and U.S. Pat. No. 7,951,579.

Any suitable phage virus or viruses, including any of those mentionedherein, can be included in the viral preparation. A viral preparationaccording to the invention is a composition containing a vector or viralmaterial with the ability to interact with one or more differenttargets. A broad-host-range can be provided by including a cocktail ofphages (e.g., two or more of any phage such as those mentioned herein),a phage that infects a range of hosts, or a combination thereof.

Any suitable broad-host-range phage can be used. For example, phageSN-1, SN-2, SN-X, SN-T, BHR1, BHR2, BHR3, BHR4, BHR5, PRD1, KVP40,PY100, PRD1, PVP-SE1, or a combination thereof may be included in aviral preparation. Broad host range phages are discussed in Miller etal., 2003, Complete genome sequence of the broad-host-range vibriophageKVP40: comparative genomics of a T4-related bacteriophage; J Bact185(17):5220-5233; Beumer, 2005, A broad-host-range, generalizedtransducing phage SN-T acquires 16S rRNA genes from different genera ofbacteria, Appl Env Microb 71(12):8301-8304; Green et al., 1985,Isolation and preliminary characterization of lytic and lysogenic phageswith wide host range within the streptomycetes, J. Gen Microbiol131(9):2459-2465; Jensen et al., 1998, Prevalence of broad-host-rangelytic bacteriophages of Sphaerotilus natans, Escherichia coli, andPseudomonas aeruginosa, Appl Environ Microbiol 64(2):575-580; Bamford etal., 1995, Bacteriophage PRD1: a broad host range dsDNA tectivirus withan internal membrane, Adv Virus Res 45:281-319; Schwudke, et al., 2008,Broad-host-range Yersinia phage PY100: genome sequence, proteomeanalysis of virions, and DNA packaging strategy, J Bact 190(1):332-342;Olsen et al., 1974, Characteristics of PRD1, a plasmid-dependent broadhost range DNA bacteriophage, J Viriol 14(3):689-699; Santos et al.,2011, Genomic and proteomic characterization of the broad host rangeSalmonella phage PVP-SE1: creation of a new phage genus, J Viriol85(21):11265-73; Sillankorva et al., Efficacy of a broad host rangelytic bacteriophage against E. coli adhered to urothelium, CurrMicrobiol 62(4):1128-1132; Evans et al, 2010, Characterization of abroad-host-range flagellum-dependent phage that mediates high-efficiencygeneralized transduction in, and between, Serratia and Pantoea,Microbiol 156:240-247; Garbe et al., 2010, Characterization of JG024, aPseudomonas aeruginosa PB1-like broad-host range phage under simulatedinfection conditions, BMC Microbiol 10:301; Schwarzer et al., 2012, Amultivalent adsorption apparatus explains the broad host range of phagephi92: a comprehensive genomic and structural analysis, J ViriolJVI.00801-12; U.S. Pub. 2012/0168372; U.S. Pub. 2012/0128652; and U.S.Pub. 2011/0064699, the contents of which are incorporated by reference.

A broad-host-range viral preparation can be provided by including morethan one phage in the composition. For example, if each included phageis specific for one species, the phage cocktail will have the ability toinfect multiple species. Any number of different phages (e.g., 3, 5,tens, hundreds) may be included in a phage cocktail. Those phages maythemselves be narrow or broad in host range. Phage cocktails arediscussed in Kelly et al., 2011, Development of a broad-host-range phagecocktail for biocontrol, Bioengineered Bugs 2:1, 31-37; Int. ApplicationPub. WO 02/07742; U.S. Pat. No. 6,121,036; U.S. Pub. 2009/0047254; andU.S. Pub. 2005/0032036, the contents of which are hereby incorporated byreference.

Other vectors may be used to cause different unknown pathogens topresent a common binding element. Suitable vectors include plasmids,phagemids, and bacterial ghosts. In some embodiments, a plasmid isincluded, optionally enveloped in a lipid layer (e.g., a liposome,micelle, or reverse micelle), bi-layer, or a protein coat. Suitableplasmids are known in the art and include Col E1, RSF1030, clo DF13,R6K, F, R1, and Ent P 307.

In certain embodiments, one or more of the pathogens are targeted usinga bacterial ghost. Bacterial ghosts are bacterial cell envelopes devoidof cytoplasmic content. A bacterial ghost can be included that exhibitsone or more cell surface moieties that bind to any number ofcell-surface targets on one or a range of bacteria. Further, thebacterial ghosts may each include the known common binding element(e.g., common antigen, biotinylated protein, streptavidin, etc.).Bacterial ghosts may be preferred since they can carry multiple surfacemoieties due to their size and are non-pathogenic themselves due toinactivity and can be stored (e.g., on-chip) for long periods of time.Bacterial ghosts are discussed in Lubitz et al., 2009, Applications ofbacterial ghosts in biomedicine, Adv Exp Med Biol 655:159-70; Langemannet al., 2010, The bacterial ghost platform system: production andapplications, Bioeng Bugs 1(5):326-36; Tabrizi et al., 2004, Bacterialghosts-biological particles as delivery systems for antigens, nucleicacids, and drugs, Curr Opp Biotechnol 15:530-537; U.S. Pat. No.7,067,639; U.S. Pat. No. 6,896,887; U.S. Pub. 2012/0040829; and U.S.Pub. 2010/0203082, the contents of which are incorporated by referencefor all purposes.

Preferably, the viral preparation is introduced 107 with a highconcentration of phage or vector so that viruses attach 113 to alltarget pathogens. Viral preparations can further include additionalcomponents to facilitate transduction by, for example, contributing tothe competency of the target cells to receive extrinsic genetic materialor to inhibit infection or lysis by sources other than the phage vectoror vectors used. Preparations may include one or more salt, buffer,chelator, ion, or combination thereof, in any suitable concentration.Infectious, non-transducing phage can be inhibited by ion chelators(e.g., citrate or EGTA). In some embodiments, bacterial nutrients areadded to the viral preparation to encourage cell vitality. For example,trypticase soy or a sugar can be included.

As shown in FIG. 1, a binding partner, optionally bound to a substrate,is obtained 125 and introduced into the sample. The sample with thebinding partner is incubated 131 so that the binding partner can bind133 to the binding element on the pathogens. In some embodiments, thevirus or vector is used to cause different pathogens to presentdifferent binding elements. The pathogens may then be extracted usingdifferent binding partners. For example, phages can be used that causesome bacteria to express antigen A, some to express antigen B, and soon. Substrate 219, such as magnetic beads, can then be used that includeanti-A, anti-B, etc., antibodies bound to the surface. The differentbinding partners may be mixed on each substrate, or separated (e.g.,some beads specific for antigen A and some for antigen B). This canfacilitate downstream sorting (e.g., where magnetic meads have differentmagnetic strengths or other sortable properties). Once the pathogens arebound to the binding partner, the pathogens can be isolated 135 from theremainder of the sample. Methods are provided to optionally concentrate139 the isolated pathogens. Isolation and concentration are discussedbelow.

The isolated pathogens may be handled according to whether isolated livecells or another product is intended 141. For live cells, the pathogensmay be introduced 147 into a live cell buffer. Any other suitabledetection or isolation may be performed. For example, where a productfrom the cells is desired, a lysis buffer may be introduced 151 to lyse155 the cells. The lysate may be purified, extracted, separated, orsimilar. In some embodiments, DNA is extracted by separation 159 on acolumn.

Methods of the invention include using a viral preparation to cause thetarget pathogens to present 119 a binding element. Any suitable methodof associating a pathogen with a binding element may be used with theinvention. Suitable methods include expression of a cell-surfaceprotein, phage display of a protein, or any other method of exposing aprotein. A protein can be a binding element such as antigen or a proteincan be further functionalized to present a binding element. For example,a protein can include a target for biotinylation and can be biotinylatedsuch that biotin is a binding element and streptavidin can be used as abinding partner.

FIG. 2 illustrates the use of a viral preparation 207 to induce theexpression of a common antigen 211 according to certain embodiments. Asshown in FIG. 2, a sample containing unknown pathogens 201 is obtained101 and viral preparation 207 is introduced 107. Viruses attach 113 topathogens 201 and introduce exogenous DNA into those cells.

Phage viruses, such as any suitable one of those discussed herein, canbe engineered to contain a gene for a common antigen 211 such as a cellsurface protein. The phage or phages from viral preparation 207 injectthe gene into pathogens 201. Pathogens 201 then express the gene causingthem to present 119 antigen 211 on their cell surface. Engineering agene into a phage vector is discussed in Green and Sambrook, 2012,Molecular Cloning: A Laboratory Manual 4Ed, Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y.), 2,000 pages and Primroseand Twyman, 2006, Principles of Gene Manipulation and Genomics, 7thEdition, Wiley-Blackwell (Malden, Mass.) 672 pages. Induction of antigenexpression is discussed in Prisco and Berardinis, 2012, Filamentousbacteriophage Fd as an antigen delivery system in vaccination, Int J.Mol. Sci. 13:5179-5194 and Gahan et al., 2009, Bacterial antigenexpression is an important component in inducing an immune response toorally administered Salmonella-delivered DNA vaccines, PLoS One4(6):e6062.

Antibody 221 may be obtained for use as the binding partner 125. Asshown in FIG. 2, antibody 221 is bound to substrate 219. Substrate 219can be any suitable substrate known in the art. In some embodiments,substrate 219 is the surface of a fluidic channel or other vessel. Insome embodiments, substrate 219 is a biomolecule, such as aoligonucleotide, a protein, an aptamer, biotin, or some other suchmoiety that itself, in turn, has affinity for another material. Forexample, substrate 219 can be a DNA or RNA sequence, optionally with oneor more locked nucleic acid (LNA) bases therein, and can be captured byexposure to a complementary sequence, such as a primer or oligo fixed ona glass slide or within a well in a microtitre plate. In certainembodiments, substrate 219 is a magnetic bead.

With continued reference to FIG. 2, the binding partner (e.g., antibody221) is introduced into the vessel containing cells 201 expressingcell-surface antigen 211 and incubated 131. Incubation of antibody-beadcomplexes with cells 201 leads to binding 133 of substrate 219 to cells201. Cells 201 can then be isolated 135 from the sample. Where substrate219 is a surface of a vessel or channel, or a surface of beads packedinto a column or otherwise held in place, cells 201 can be isolated 135by using a wash solution to wash away excess sample. Where substrate 219is a magnetic bead, a magnet 231 can then be used to isolate 135 cells201 from the remainder of the sample.

Magnetic materials may be bound to target entities and used to separatesuch entities through the use of magnet fields and gradients. Magneticmaterials and separations are known in the art and have been previouslydescribed, for example, in Murphy, 2011, Janeway's Immunobiology 8 Ed,Garland Science (New York, N.Y.), 888 pages, the contents of which areincorporated by reference herein. Methods of producing suitable magneticparticles are known in the art. See for example U.S. Pat. No. 5,597,531;U.S. Pat. No. 4,230,685; U.S. Pat. No. 4,677,055; U.S. Pat. No.4,695,393; U.S. Pat. No. 5,695,946; U.S. Pat. No. 4,018,886; U.S. Pat.No. 4,267,234; U.S. Pat. No. 4,452,773; U.S. Pat. No. 4,554,088; U.S.Pat. No. 4,659,678; U.S. Pat. No. 5,186,827; U.S. Pat. No. 4,795,698,the contents of each of which are incorporated by reference.

Any type of magnetic particle may be used for substrate 219 inaccordance with the invention. Methods of the invention include use ofdiamagnetic materials, paramagnetic materials, superparamagneticmaterials, ferromagnetic materials, or a combination thereof(independently or in combination). Diamagnetic materials are slightlyrepelled by a magnetic field and the material does not retain themagnetic properties when the external field is removed. Paramagneticmaterials (e.g., aluminum or platinum) are slightly attracted by amagnetic field and the material does not retain the magnetic propertieswhen the external field is removed. Superparamagnetic materials are muchmore susceptible to magnetization than paramagnetic materials. SeeGittleman et al., 1974, Superparamagnetism and relaxation effects ingranular Ni—SiO₂ and Ni—Al₂O₃ films, Phys Rev B 9:3891-3897.Ferromagnetic materials (e.g., iron or nickel) exhibit a strongattraction to magnetic fields and are able to retain their magneticproperties after the external field has been removed.

Further, magnetic properties of substrate 219 may depend on the size ofthe particles of substrate. Some ferromagnetic materials, e.g., magneticiron oxide, may be characterized as superparamagnetic when provided incrystals of about 30 nm or less in diameter. Larger crystals offerromagnetic materials, by contrast, retain permanent magnetcharacteristics after exposure to a magnetic field and tend to aggregatethereafter due to strong particle-particle interaction. In certainembodiments, the particles are superparamagnetic particles. In certainembodiments, the magnetic particle is an iron containing magneticparticle. In other embodiments, the magnetic particle includes ironoxide or iron platinum.

In some embodiments, the magnetic particles for substrate 219 include aportion of super-paramagnetic particles by weight (e.g., 20%, 40%, 60%,or 80%). Particles 219 can have a diameter between about 100 nm andabout 1,000 nm. In a particular embodiment, substrate 219 includessuperparamagnetic particles of diameter between about 100 nm and about250 nm. See discussion in U.S. Pub. 2011/0262989, the contents of whichare incorporated by reference.

In certain embodiments, the particles are particles (e.g.,nanoparticles) that incorporate magnetic materials, or magneticmaterials that have been functionalized, or other configurations as areknown in the art. In certain embodiments, nanoparticles may be used thatinclude a polymer material that incorporates magnetic material(s), suchas nanometal material(s). When those nanometal material(s) orcrystal(s), such as Fe₃O₄, are superparamagnetic, they may provideadvantageous properties, such as being capable of being magnetized by anexternal magnetic field, and demagnetized when the external magneticfield has been removed. This may be advantageous for facilitating sampletransport into and away from an area where the sample is being processedwithout undue particle aggregation.

One or more or many different nanometal(s) may be employed, such asFe₃O₄, FePt, or Fe, in a core-shell configuration to provide stability,and/or various others as may be known in the art. In many applications,it may be advantageous to have a nanometal having as high a saturatedmoment per volume as possible, as this may maximize gradient relatedforces, and/or may enhance a signal associated with the presence of theparticles. It may also be advantageous to have the volumetric loading ina particle be as high as possible, for the same or similar reason(s). Inorder to maximize the moment provided by a magnetizable nanometal, acertain saturation field may be provided. For example, for Fe₃O₄superparamagnetic particles, this field may be on the order of about 0.3tesla (T).

The size of the nanometal-containing particle may be optimized for aparticular application, for example, maximizing moment loaded upon atarget, maximizing the number of particles 219 on a target 201 with anacceptable detectability, maximizing desired force-induced motion,and/or maximizing the difference in attached moment between the labeledtarget and non-specifically bound targets or particle aggregates orindividual particles. While maximizing is referenced by example above,other optimizations or alterations are contemplated, such as minimizingor otherwise desirably affecting conditions.

In an exemplary embodiment, a polymer particle containing 80 wt % Fe₃O₄superparamagnetic particles, or for example, 90 wt % or highersuperparamagnetic particles, is produced by encapsulatingsuperparamagnetic particles with a polymer coating to produce a particlehaving a diameter of about 250 nm.

Each set of magnetic particles 219 has a binding partner 221 that allowsfor each set to specifically bind 133 the target 201 of interest in thesample. The binding partner may be any molecule known in the art andwill depend on the target to be captured and isolated. Exemplary bindingpartners include nucleic acids, proteins, ligands, antibodies, aptamers,and receptors.

In particular embodiments, the binding partner 221 is an antibody, suchas an antibody that binds a particular antigen 211 (see, e.g., FIG. 2).General methodologies for antibody production, including criteria to beconsidered when choosing an animal for the production of antisera, aredescribed in Kontermann, 2010, Antibody Engineering Volume 1 2Ed,Springer-Verlag (Berlin Heidelberg) 800 pages, and Harlow, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y.) 726 pages. For example, an animal of suitablesize such as goats, dogs, sheep, mice, or camels are immunized byadministration of an amount of immunogen, such the target bacteria,effective to produce an immune response. An exemplary protocol is asfollows. The animal is injected with 100 milligrams of antigenresuspended in adjuvant, for example Freund's complete adjuvant,dependent on the size of the animal, followed three weeks later with asubcutaneous injection of 100 micrograms to 100 milligrams of immunogenwith adjuvant. Additional doses are administered until a suitable titerof antibody in the animal's blood is achieved. Exemplary titers includea titer of at least about 1:5000 or a titer of 1:100,000 or more, i.e.,the dilution having a detectable activity. The antibodies are purified,for example, by affinity purification on columns containing protein Gresin or target-specific affinity resin.

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Such techniques are known in the art.See, e.g., Mulder et al., 1993, Characterization of two human monoclonalantibodies reactive with HLA-B12 and HLA-B60, respectively, raised by invitro secondary immunization of peripheral blood lymphocytes, Hum.Immunol 36(3):186-192; Stauber et al., 1993, Rapid generation ofmonoclonal antibody-secreting hybridomas against African horse sicknessvirus by in vitro immunization and the fusion/cloning technique, JImmunol Methods 161(2):157-168; and Venkateswaran, et al., 1992,Production of anti-fibroblast growth factor receptor monoclonalantibodies by in vitro immunization, Hybridoma, 11(6):729-739. Thesetechniques can be used to produce antigen-reactive monoclonalantibodies, including antigen-specific IgG, and IgM monoclonalantibodies.

Any antibody or fragment thereof having affinity and specific for thetarget of interest may be used for binding partner 221 within the scopeof the invention provided herein.

Methods for attaching the binding partner 221 to the magnetic particle219, including the coating of particles with antibodies, are known inthe art. See for example Kontermann, 2010, Antibody Engineering Volume 12Ed, Springer-Verlag (Berlin Heidelberg) 800 pages; Harlow, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y.) 726 pages; and Stanley, 2002, Essentials inImmunology and Serology, Delmar Cengage (Independence, Ky.) 560 pages.Such methodology can easily be modified by one of skill in the art tobind other types of binding partners to the magnetic particles. Inaddition, certain types of magnetic particles coated with a functionalmoiety are commercially available from Sigma-Aldrich (St. Louis, Mo.).

In some embodiments, more than one antibody 221 is used to create setsof magnetic particles. Since each set of particles 219 may be conjugatedwith antibodies 221 having different specificities for one or moreantigen 211 coded in the genome of the phage or phages, compositions andconcentrations may be optimized for detection of multiple unknownpathogens in the sample. In certain embodiments, all of the sets areprovided at the same concentration. Alternatively, the sets are providedat different concentrations. For example, compositions may be designedsuch that sets that bind gram positive bacteria (e.g., antibody 221 toantigen 211 in genome of phage that targets Gram + cells 201) are addedto the sample at a concentration of 2×10⁹ particles per/ml, while setsthat bind gram negative bacteria (e.g., antibody to antigen in genome ofphage specific to Gram −) are added to the sample at a concentration of4×10⁹ particles per/ml. Compositions used with methods of the inventionare not affected by antibody cross-reactivity. However, in certainembodiments, sets are specifically designed such that there is nocross-reactivity between different antibodies and different sets.

To facilitate downstream detection of pathogens 201, one or more labelsmay be added to antibody 221, particle 219, or both. For example, alabel may be added during preparation of antibody/bead complexes. Anydetectable label may be used with compositions of the invention, such asfluorescent labels, radiolabels, enzymatic labels, and others. Thedetectable label may be directly or indirectly detectable. In certainembodiments, the exact label may be selected based, at least in part, onthe particular type of detection method used. Exemplary detectionmethods include radioactive detection, optical absorbance detection(e.g., UV-visible absorbance detection, optical emission detection,e.g., fluorescence, phosphorescence, chemiluminescence), or Ramanscattering. Preferred labels include optically-detectable labels, suchas fluorescent labels. Examples of fluorescent labels include withoutlimit acridine and derivatives; BODIPY; Brilliant Yellow; coumarin andderivatives; DABITC; eosin and derivatives; erythrosin and derivatives;isothiocyanate; ethidium; fluorescein and derivatives; FAM; DTAF; QFITC,(XRITC); Malachite Green isothiocyanate; Phenol Red; pyrene andderivatives; Reactive Red 4 (Brilliant Red 3B-A sold under the trademarkCIBACRON); rhodamine and derivatives; Texas Red; TAMRA; TRITC;riboflavin; Atto dyes; La Jolta Blue; phthalo cyanine; and naphthalocyanine. Preferred labels include cyanine-3 and cyanine-5. Labels otherthan fluorescent labels are contemplated by the invention, includingother optically-detectable labels. Methods of linking fluorescent labelsto magnetic particles or antibodies are known in the art. Suitablelabels and methods of their use are described in U.S. Pub. 2011/0262926,the contents of which are hereby incorporated by reference.

As shown in FIG. 2, the magnetic particles described above are thenintroduced 125 to the sample in order to bind 133 to the unknownpathogens 201. For example, a blood sample may be mixed with thedescribed magnetic particles to generate a mixture that is allowed toincubate 131 such that the compositions bind to at least one pathogen201 in the blood sample. The mixture is allowed to incubate 131 for asufficient time to allow for the composition to bind to the pathogen inthe blood. The process of binding the composition to the pathogenassociates a magnetic moment with the pathogen, and thus allows thepathogen to be manipulated through forces generated by magnetic fieldsupon the attached magnetic moment.

In general, time for incubation 131 will depend on the desired degree ofbinding between the pathogen and the compositions of the invention(e.g., the amount of moment that would be desirably attached to thepathogen), the amount of moment per target, the amount of time ofmixing, the type of mixing, the reagents present to promote the bindingand the binding chemistry system that is being employed. Incubation timecan be anywhere from about 5 seconds to a few days. Exemplary incubationtimes range from about 10 seconds to about 2 hours. Binding occurs overa wide range of temperatures, generally between about 5° C. and about65° C., e.g., between about 15° C. and about 40° C.

In certain embodiments, a buffer solution is added to the sample alongwith the compositions of the invention. An exemplary buffer includesTris(hydroxymethyl)-aminomethane hydrochloride (Tris HCl) at aconcentration of about 75 mM. It has been found that the buffercomposition, mixing parameters (speed, type of mixing, such as rotation,shaking etc., and temperature) influence binding. It is important tomaintain osmolality of the final solution (e.g., blood+buffer) tomaintain high label efficiency. In certain embodiments, buffers used inmethods of the invention are designed to prevent lysis of blood cells,facilitate efficient binding of targets with magnetic particles and toreduce formation of particle aggregates. It has been found that thebuffer solution containing 300 mM NaCl, 75 mM Tris-HCl pH 8.0 and 0.1%Tween 20 meets these design goals.

Without being limited by any particular theory or mechanism of action,it is believed that sodium chloride is mainly responsible formaintaining osmolality of the solution and for the reduction ofnon-specific binding of magnetic particle through ionic interaction.Tris HCl is frequently used in biology to maintain pH of a solution. Ithas been found that 75 mM concentration is beneficial and sufficient forhigh binding efficiency. Likewise, Tween 20 is widely used as a milddetergent to decrease nonspecific attachment due to hydrophobicinteractions. Various assays use Tween 20 at concentrations ranging from0.01% to 1%. The 0.1% concentration appears to be optimal for theefficient labeling of bacteria, while maintaining the integrity of bloodcells.

Additional compounds can be used to modulate the capture efficiency byblocking or reducing non-specific interaction with blood components andeither magnetic particles or pathogens. For example, chelatingcompounds, such as EDTA or EGTA, can be used to prevent or minimizeinteractions that are sensitive to the presence of Ca²⁺ or Mg²⁺ ions.

One can also use a static mixer or other mixing device that providesefficient mixing of viscous samples at high flow rates to achieve highbinding efficiency while reducing time required for the binding step. Inone embodiment, the sample is mixed with binding buffer in ratio of, orabout, 1:1, using a mixing interface connector. The diluted sample thenflows through a mixing interface connector where it is mixed withtarget-specific nanoparticles. Additional mixing interface connectorsproviding mixing of sample and antigen-specific nanoparticles can beattached downstream to improve binding efficiency. The combined flowrate of the labeled sample is selected such that it is compatible withdownstream processing.

With reference to FIG. 2, after binding 133 of particles 219 topathogens 201 in the sample to form pathogen/magnetic particlecomplexes, a magnetic field 231 may be applied to the mixture to captureor isolate 135 the complexes on a surface. Components of the mixturethat are not bound to magnetic particles will not be affected by themagnetic field and will remain free in the mixture. Methods andapparatuses for separating 135 target/magnetic particle complexes fromother components of a mixture are known in the art. For example, a steelmesh may be coupled to a magnet, a linear channel or channels may beconfigured with adjacent magnets, or quadrapole magnets with annularflow may be used. Other methods and apparatuses for separatingtarget/magnetic particle complexes from other components of a mixtureare shown in U.S. Pat. No. 7,699,979; U.S. Pat. No. 6,551,843; U.S. Pat.No. 5,622,831; U.S. Pat. No. 6,514,415; U.S. Pat. No. 5,695,946; U.S.Pat. No. 5,186,827; U.S. Pat. No. 5,541,072; U.S. Pat. No. 5,466,574;and U.S. Pat. No. 6,623,983, the contents of each of which areincorporated by reference.

In certain embodiments, the magnetic capture is achieved at highefficiency by utilizing a flow-through capture cell with a number ofstrong rare earth bar magnets 231 placed perpendicular to the flow ofthe sample. When using a flow chamber with flow path cross-section 0.5mm×20 mm (h×w) and 7 bar NdFeB magnets, the flow rate could be as highas 5 mL/min or more, while achieving capture efficiency close to 100%.

The foregoing illustrative embodiment is described generally in terms ofthe induction of the expression of a common antigen 211 by one or moredifferent unknown pathogen 211, as shown in FIG. 2. The inventiongenerally provides methods of using a vector or viral preparation 207 tocause unknown pathogens 201 to present a common binding element.

FIG. 3, for example, illustrates an alternative embodiment of a methodof using a viral preparation 207 to cause different unknown pathogens topresent 119 a common binding element 303. As shown in FIG. 3, viralpreparation 207 includes at least one phage.

In certain embodiments, a bacteriophage is used that expresses a biotinbinding domain (aka, a biotinylation peptide) on at least one capsidprotein. The phage contains the genetic information to display abiotinylation domain and be biotinylated. In some embodiments, the phagedisplays a biotinylation domain when introduced 107 into the sample.When the phage attach to cells 201, those cells are then biotinylated.In certain embodiments, the phage infects the target cells and progenyvirions produced from the infection will have been biotinylated by thetarget cells' 201 biotin ligase protein (BLP). Biotinylation of phagewith biotinylation peptides on their capsids is discussed in Edgar etal., 2006, High-sensitivity bacterial detection using biotin-taggedphage and quantum-dot nanocomplexes, PNAS 103(13):4841-4945 and Gerviaset al., 2007, Immobilization of biotinylated bacteriophages on biosensorsurfaces, Sensors and Actuators B 125:615-621, each of which areincorporated by reference. Methods of labeling targets, e.g., includingbiotinylation of phage, are discussed in U.S. Pat. No. 7,517,643; U.S.Pat. No. 6,342,588; and U.S. Pub. 2011/0086338, the contents and methodsof each are incorporated by reference in their entirety for allpurposes. In some embodiments, phage capsid is streptavidin oravidin-linked and substrate 219 is biotinylated, as discussed in U.S.Pat. No. 6,740,492, the contents of each are incorporated by referencein their entirety for all purposes.

With continued reference to FIG. 3, cells 201 present 119 biotin 303 asa common binding element. Binding partner 125 is introduced. Here,binding partner 125 includes streptavidin 309 coated beads 219.Biotinylation of phage is further discussed in Smelyanski and Gershoni,2011, Site directed biotinylation of filamentous phage structuralproteins, Virol J 8:495; U.S. Pat. No. 5,994,519; and U.S. Pub.2001/0019820, the contents and methods of each are incorporated byreference in their entirety for all purposes.

Biotinylated target cells 201 are incubated 131 with streptavidin-coatedbeads 219. The binding partner binds 133 to the target, after which thetarget 201 can be isolated 135. For example, where beads 219 aremagnetic, a magnet 231 can be used to hold cells 201 in a fluidicchannel while the sample is washed away. With reference back to FIG. 1,where live cells are intended 139, the sample can be replaced with amaintenance buffer 147. In an alternative embodiment, cells 201 can belysed 155, optionally using a lysis buffer 151. In some embodiments, acomponent of the cells 201 is extracted. For example, DNA may optionallybe extracted by column separation 159.

While discussed above in the illustrative embodiments involvingbiotinylated phage, streptavidin-linked phage, or expression of a commonantigen after transduction by a phage, methods of the invention includeany suitable method or phenomenon to cause different unknown pathogens201 to exhibit a common binding element through the introduction of aviral preparation 207. In some embodiments, phage display of an antigenis employed.

FIG. 4 illustrates phage display of a common antigen by differentunknown pathogens 201. In phage display, a gene for antigen 411 isligated into the phage genome, for example, within the gene encoding oneof the coat proteins, or capsid proteins. Multiple cloning sites may beused to ensure in-frame cloning and proper expression of the proteinproduct. The viral preparation 207 including one or more phageengineered for phage display of a common antigen 411 is then introduced107 into the sample containing unknown pathogens 201. A binding partneris used (e.g., linked to a substrate 219 such as a well in a microtitreplate, a surface of a fluidic channel, beads in a column, or magneticbeads) that binds to antigen 411. After the phage attach 119 topathogens 201, they display antigen 411. Phage display is discussed inHaq, 2012, Bacteriophages and their implications on futurebiotechnology, a review, Virol J 9:9; U.S. Pat. No. 8,227,242 (e.g.,phage display without helper phage); U.S. Pat. No. 8,216,797; U.S. Pat.No. 7,238,669; U.S. Pat. No. 6,740,492; U.S. Pub. 2010/0240579(detection of unknown target by phage display); and U.S. Pub.2006/0063149, the contents of which are incorporated by reference.

In some embodiments, phage display antigen 411 when introduced 107 intothe sample. In certain embodiments, the genetically-engineered phage isintroduced and infects pathogens 201 and generates progeny virions thatexpress antigen 411. Viral preparation 207 may include a bacterialgrowth medium to allow low concentrations (e.g., single cells) of targetto grow and be infected and propagate the phage. Moreover, a combinationof the foregoing may occur in that phage may exhibit antigen 411 uponintroduction 107 into the sample, and may infect and propagate,generating progeny.

While any suitable substrate 219 may be used, and any suitable binder(e.g., aptamers, co-factors, proteins, etc.), in certain embodiments,magnetic beads linked to antibody 221 are used. This binding partner 125is introduced into the infected sample and allowed to incubate 131.

FIG. 3 shows that incubation 131 leads to the binding 133 of bindingpartner 125 to antigen 411 on cells 201. After cells 201 are bound tomagnetic beads 219 via antibody 221/antigen 411 interaction, the cells201 may be isolated 135 from the sample by magnetic separation. Further,additional steps may be performed to optimize the isolation orextraction of cells 201 from the sample. Additional steps optionallyinclude washing with one or more solutions, further concentrating 139captured cells (e.g., using a magnetic concentrator), introduction ofadditional buffers such as, for example, cell maintenance buffers orlysis buffers, other suitable processes known in the art, or acombination thereof.

Other methods of using a binding partner to capture a target areoperable with methods of the invention. For example, where pathogens 201are intracellular parasites, a known binding element may be associatedwith the host cells. A binding partner may be used that would not beable to target an unknown pathogen directly but instead bind to the host(if the host is eukaryotic, instead of a phage viral preparation, abinding partner specific to a eukaryotic protein may be employed). See,e.g., Drancourt et al., 1992, Diagnosis of Mediterranean spotted feverby indirect immunofluorescence of Rickettsia conorii in circulatingendothelial cells isolated with monoclonal antibody-coatedimmunomagnetic beads, J Inf Dis 166:660-3, incorporated by reference.Optionally, the membrane of the eukaryotic host can be ruptured (e.g.,with detergent, a virus or particle, differential osmolality, etc.,) andthe infectious agent targeted by methods herein. In some embodiments,methods of the invention may be used in combination with, or in sequencewith, alternative methodologies that would only operate, for example,where one or more target is known. If a target is known, it may exhibita known binding element that can be captured with a binding partner.See, e.g., Fu et al., Rapid detection of E. coli O157:H7 byimmunomagnetic separation and real-time PCR, incorporated by reference.

The process of magnetic separation 135 described above (e.g., withrespect to FIG. 3) produces efficient capture of different unknownpathogens 201 and the removal of all or majority of the remainingcomponents in the mixture. However, it is still possible that arelatively small amount of non-specific analytes are unintentionallycaptured along with the target/magnetic particle complexes. It may bedesired to remove these non-specific analytes. Accordingly, the surfacemay be washed with a wash solution that reduces particle aggregation,thereby isolating target/magnetic particle complexes from thenon-specific target entities.

Any wash solution that does not disrupt interaction between the bindingpartner of the magnetic particle and the target may be used. The washsolution should also not disrupt the capture of the target/particlecomplexes on the intended surface. Exemplary solutions include heparin,Tris-HCl, phosphate buffered saline (PBS), Tris-borate-EDTA (TBE),Tris-acetate-EDTA (TAE), Tris-cacodylate, HEPES, or similar. One or morewash cycle may be performed. For embodiments in which the sampleincludes blood, heparin may be used to inhibit clotting. The boundtargets are washed with heparin-containing buffer 1-3 times to removeblood components and to reduce formation of aggregates. Methods forthese steps are described in U.S. Pat. No. 7,776,580; U.S. Pub.2011/0263833; U.S. Pub. 2011/0262989; U.S. Pub. 2011/0262933; U.S. Pub.2011/0262927; U.S. Pub. 2011/0262893; and U.S. Pub. 2010/0092956, thecontents of which are incorporated by reference for all purposes.

Once the different unknown pathogens 201 have been isolated 135, theymay be preserved as living cells or they may be lysed and the lysate, ora component thereof, may be detected, extracted, isolated, quantified,or used.

Where live cells are intended 141, a live cell buffer may be introduced147, for example, in a step that includes flushing away any washbuffers, other solutions or reagents, or remaining components of thesample.

Once the target/magnetic particles have been captured, the target isthen lysed 155. In certain embodiments, lysis of the target occurswithout separating the particles from the target prior to the lysisstep. Conducting the lysis without the pre-separation step allows moreefficacious collection of analytes contained within the target. Forinstance, if the analyte of interest is a bacterially-derived nucleicacid, some analyte may be lost when bacteria separated from the magneticparticles are inadvertently lost. With the disclosed methods, thebacteria are still bound to the particles and captured on a surface aslysis occurs. Accordingly, there is a concentrated sample to work withduring the lysis step. In addition, the disclosed methods allow forrecovery of a specific analyte in a relatively small collection volume.This is especially useful when the analyte of interest is a nucleic acidor something that is similarly present in only very small quantities.For example, one could begin with a blood sample of 2 ml and use thedescribed methods to concentrate a desired pathogen from the blood ontoan appropriate surface. With the pathogen captured, one could decant theblood, add 0.3 ml of a suitable buffer, and subsequently perform thelysis step.

Lysis 155 can be performed using any means known in the art. Forexample, lysis could be performed using 151 a lysis buffer (see, e.g.,FIG. 1). Any type of lysis buffer is suitable for use with the disclosedmethods; selection of the specific buffer may depend on the subsequentanalysis of the cell lysate. Buffer selection is within the generalskill of the art and can be determined empirically. Generally, lysisbuffers contain Tris-HCL, EDTA, EGTA, SDS, deoxycholate, Triton X,and/or NP-40. In some cases the buffer may also contain NaCl (e.g., 150mM). In certain aspects, the lysis buffer is a chaotropic solution.

Lysis 155 can also be achieved through, or assisted by, sonication. Inthis method, the captured target/magnetic particle complexes are exposedto ultrasonic waves to achieve lysis of any target (bacteria, cells,virus, fungi, etc.) associated with the magnetic particles so that anyanalytes of interest contained therein are released. In someembodiments, the analyte of interest can include a nucleic acid.

The methods described herein can be used in accordance with anysonication device, which are well-known in the art. In certainembodiments, the sonication device is the VCX 750 Sonicator sold underthe trademark VIBRA-CELL (sonicator, commercially available from Sonicsand Materials, Inc.). Generally, the probe of the sonicator is placedinto the liquid containing the targets to be lysed. Electrical energyfrom a power source is transmitted to a piezoelectric transducer withinthe sonicator converter, where it is changed to mechanical vibrations.The longitudinal vibrations from the converter are intensified by theprobe, creating pressure waves in the liquid. These in turn producemicroscopic bubbles, which expand during the negative pressure excursionand implode violently during the positive excursion. This phenomenon,referred to as cavitation, creates millions of shock waves and releaseshigh levels of energy into the liquid, thereby lysing the target. Inanother embodiment, the sonication transducer may be brought in contactwith a chamber holding captured complexes by way of a structuralinterface. The sonication transducer vibrates the structural interfacesuch that lysis is achieved. In either method, the appropriate intensityand period of sonication can be determined empirically by those skilledin the art.

The lysate containing the contents of the lysed target can then beeluted. In certain aspects, the lysate contains nucleic acids ofinterest associated with a particular bacteria present in the startingsample. The lysate is removed from the magnetic particles and theanalytes contained therein can be analyzed. Analytes may include,without limitation, nucleic acids, proteins, organelles, and othercomponents found within the target of interest.

The analyte may be analyzed by a multitude of existing technologies,such as NMR, Polymerase Chain Reaction (PCR), sequencing, massspectrometry, fluorescent labeling and visualization using microscopicobservation, fluorescent in situ hybridization (FISH), growth-basedantibiotic sensitivity tests, and variety of other methods that may beconducted with purified target without significant contamination fromother sample components. Analysis using NMR is described in U.S. Pub.2011/0262925, herein incorporated by reference in its entirety. In someembodiments, the different unknown pathogens 201 are analyzed in orderto identify genes they express. For example, captured bacteria 201 arelysed without first separating the bacteria from the magnetic particles.The lysate is then eluted from the magnetic particles and DNA containedwithin the lysate/eluate is bound to DNA extraction resin. After washingof the resin, the bacterial DNA is eluted and used in quantitativeRT-PCR to detect the presence of a specific species, and/or, subclassesof bacteria.

Detection of bacteria of interest can be performed by use of nucleicacid probes following procedures which are known in the art. Suitableprocedures for detection of bacteria using nucleic acid probes aredescribed in U.S. Pat. No. 7,943,346; U.S. Pat. No. 5,620,847; U.S. Pat.No. 5,569,586; U.S. Pat. No. 5,541,308; U.S. Pat. No. 5,401,631; U.S.Pat. No. 5,089,386; and U.S. Pat. No. 5,055,394, each of which is herebyincorporated by reference.

A suitable nucleic acid probe assay generally includes sample treatmentand lysis, hybridization with selected probe(s), hybrid capture, anddetection. Lysis 155 of the bacteria is necessary to release the nucleicacid for the probes. In accordance with the invention, a means forlysing the bacteria is introduced while the bacteria are still bound tothe magnetic particle and the resulting complexes have been captured ona surface. There is no separation of the bacteria from the magneticparticles prior to lysis. In some embodiments, the nucleic acid targetmolecules are released by treatment with any of a number of lysisagents, including alkali (such as NaOH), guanidine salts (such asguanidine thiocyanate), enzymes (such as lysozyme, mutanolysin andproteinase K), and detergents. In other embodiments, sonication is usedto lyse the cells. Lysis of the bacteria, therefore, releases both DNAand RNA, particularly ribosomal RNA and chromosomal DNA both of whichcan be utilized as the target molecules with appropriate selection of asuitable probe. Use of rRNA as the target molecule(s), may beadvantageous because rRNAs constitute a significant component ofcellular mass, thereby providing an abundance of target molecules. Theuse of rRNA probes also enhances specificity for the bacteria ofinterest, that is, positive detection without undesirablecross-reactivity which can lead to false positives or false detection.

Hybridization includes addition of the specific nucleic acid probes. Ingeneral, hybridization is the procedure by which two partially orcompletely complementary nucleic acids are combined, under definedreaction conditions, in an anti-parallel fashion to form specific andstable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mis-pairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique nucleic acid sequences of the target organism. In general, afirst capture probe is utilized to capture formed hybrid molecules. Thehybrid molecule is then detected by use of antibody reaction or by useof a second detector probe which may be labeled with a radioisotope(such as phosphorus-32) or a fluorescent label (such as fluorescein) orchemiluminescent label.

Detection of bacteria of interest can also be performed by use of PCRtechniques. A suitable PCR technique is described, for example, inVerhoef et al. (WO 92/08805). Bacterially-derived nucleic acids isolatedfrom the lysate can be used as templates for the PCR reaction. PCR canalso include the use of reverse-transcriptase PCR (RT-PCR), in which RNAisolated from the target is reverse transcribed into its DNA complement(i.e., cDNA) using the enzyme reverse transcriptase, and the resultingcDNA is amplified using PCR. RT-PCR is described in detail in U.S. Pub.2011/0071033, incorporated by reference herein in its entirety.

In certain embodiments, differential gene expression associated with thetarget can also be identified, or confirmed using a microarraytechnique. In this method, polynucleotide sequences of interest(including cDNAs and oligonucleotides) are plated, or arrayed, on amicrochip substrate. The arrayed sequences are then hybridized withspecific DNA probes from cells or tissues of interest. Methods formaking microarrays and determining gene product expression (e.g., RNA orprotein) are shown in U.S. Pub. 2006/0195269, the content of which isincorporated by reference herein in its entirety.

In particular aspects, nucleic acids isolated from the target can besequences to generate a plurality of sequence reads, thereby identifyinga pathogen according to a known genetic sequence. Sequencing may be byany method known in the art. DNA sequencing techniques include classicdideoxy sequencing reactions (Sanger method) using labeled terminatorsor primers and gel separation in slab or capillary, sequencing bysynthesis using reversibly terminated labeled nucleotides,pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allelespecific hybridization to a library of labeled oligonucleotide probes,sequencing by synthesis using allele specific hybridization to a libraryof labeled clones that is followed by ligation, real time monitoring ofthe incorporation of labeled nucleotides during a polymerization step,polony sequencing, and SOLiD sequencing. Sequencing of separatedmolecules has more recently been demonstrated by sequential or singleextension reactions using polymerases or ligases as well as by single orsequential differential hybridizations with libraries of probes. Methodsof sequencing are discussed in U.S. Pat. No. 8,209,130 and U.S. Pub.2011/0071033, the content of which is incorporated by reference hereinin its entirety.

In certain embodiments, methods of the invention are useful for directdetection of unknown bacteria 201 from blood. Such a process isdescribed here. Sample is collected in sodium heparin tube byvenipuncture, acceptable sample volume is about 1 mL to 10 mL. Sample isdiluted with binding buffer and superparamagnetic particles havingbinding partners are added to the sample, followed by incubation on ashaking incubator at 37° C. for about 30 min to 120 min. Alternativemixing methods can also be used. In a particular embodiment, sample ispumped through a static mixer, such that reaction buffer and magneticparticles are added to the sample as the sample is pumped through themixer. This process allows for efficient integration of all componentsinto a single fluidic part, avoids moving parts and separate incubationvessels and reduces incubation time.

Capture of the labeled targets allows for the removal of bloodcomponents and reduction of sample volume from 30 mL to 5 mL. Thecapture is performed in a variety of magnet/flow configurations. Incertain embodiments, methods include capture in a fluidic cartridge orother flow-through device at flow rate of 5 mL/min, resulting in totalcapture time of 6 min.

After capture, the captured target is washed with wash buffer includingheparin to remove blood components and free particles. The compositionof the wash buffer is optimized to reduce aggregation of free particles,while maintaining the integrity of the particle/target complexes.

After the wash step, the still captured targets are sonicated in orderto lyse the cells. The bacterial cells are not separated from themagnetic particles prior to sonication. While the appropriate settingsfor sonication can be determined empirically, a sonicator can be usedfor a duration of 3 minutes to achieve effective lysis. The resultinglysate can then be eluted and the expression profile of any nucleic acidfound in the lysate can be determined.

Devices for conducting the above methods are also provided. Any suitabledevice or system may be used. In general, the devices comprise an inputchannel, an output channel, a chamber, a magnetic assembly, and a lysingdevice. The input channel and output channel are in fluid communicationwith the chamber and the magnetic assembly is adapted to capture amagnetic particle inputted into the input channel onto a surface of thechamber. The lysing device is adapted to lyse a target bound to themagnetic particle. In one illustrative embodiment, the device is afluidic cartridge.

FIG. 5 illustrates a fluidic cartridge 501 according to certainembodiments. Fluidic cartridge 501 is provided to operate with bloodcollection tube 505 (e.g., a vacutainer). Cartridge 501 includes longneedle 513 that penetrates into an interior of tube 505 when tube 505 isinserted thereon. Magnetic beads are stored in magnetic bead ampule 507shown disposed within bead buffer 509. Pneumatic interface 511 providespressure to flow sample, solutions, and buffers through channels ofcartridge 501 and may optionally be used to crush ampules such asmagnetic bead ampule 507 (and plant pathogen ampule 529). When operationis begun and tube 505 containing a sample is inserted, magnetic beadampule 507 is crushed, introducing beads 219 to bead buffer 509. Beads219 and buffer 509 may be mixed, for example, using agitation providedby air from pneumatic interface 511, to produce a bead mixture.

Air from pneumatic interface 511 forces blood from tube 505 into mixingchamber 521. The bead mixture is forced through long needle 513 to rinsethe inside of tube 505. The bead mixture is then sent to mixing chamber521 to mix with the blood. This step may be repeated, sending more beadmixture from bead buffer 509, through tube 505, to mixing chamber 521,until tube 505 is evacuated of target and a desired proportion (e.g.,2:1) of bead mixture to sample is present in mixing chamber 521.

Mixing paddle 525 is used to mixing the contents of chamber 521,agitating the blood and beads. Air pressure from pneumatic interface 511then forces the mixture into magnetic trap 531. Optionally, the mixturecan be pushed back, from magnetic trap 531 to mixing chamber 521, andthe cycle repeated any number times until mixing chamber 521 issatisfactorily evacuated of target pathogens 201 and the bead-boundtarget pathogens 201 as well as the other components of the sample arein magnetic trap 431.

Then, magnetic trap 431 is evacuated of the other components of thesample, leaving magnetic particles 219 bound to magnets therein. Thewaste fluid is pushed back through mixing chamber 521 and discarded.Wash buffer 519 is then forced into magnetic trap 531, filling it. Thewash buffer can then be pushed back to mixing chamber 521 to ensure goodwashing of beads 219. Wash buffer is sent back into magnetic trap 531and held there.

Magnets can then be moved away from magnetic trap 531, allowing beads219 to re-suspend in wash buffer 519 within trap 531. At this point,target pathogens 201 have been extracted from the original sample andheld in wash buffer 519. Wash buffer 519 can then be pushed throughmagnetic concentrator 545 to waste chamber 549.

Turning now to the inset portion of FIG. 5, magnetic concentrator 545can be seen to be in fluid communication with magnetic trap 531. As washbuffer 519 flows through concentrator 545, beads 219 are captured inconcentrator 545 while the remainder of buffer 519 is passed on to wastechamber 549. Original pathogens 201 are thus concentrated inconcentrator 545.

The contents of concentrator 545 may be processed according to whetherlive cells or an extracted cellular component is intended. If live cellsare intended, the magnet is removed from magnetic concentrator 545 andbuffer 551 (here, a live cell buffer) is introduced through concentrator545 and used to flush the cells into output vial 589.

If, for example, extracted DNA is intended, buffer 551 is a lysis bufferand is introduced into magnetic concentrator 545. The magnet is removedfrom concentrator 545 and a sonicator may be applied to a wall of thechamber of concentrator 545. Optionally, beads may be included forbead-bashing to aid in lysis. The sonicator or other lysis means isactivated and the target cells 201 are lysed. In certain embodiments, aprobe of a sonicator extends into the chamber, where it deliversvibrations into the liquid medium surrounding the captured complexes. Inother embodiments, the sonication transducer is brought in contact withthe chamber by way of a structural interface. For example, thestructural interface may constitute the floor or the ceiling of thechamber. The sonication transducer vibrates the structural interfacesuch that lysis of the targets captured in the chamber is achieved.

Lysate is then pushed into pre-column mixer 557. DNA binding buffer 559is added to pre-column mixer 557 (optionally agitated by bubbling air).The contents of pre-column mixer 557 is then forced through the DNAextraction column 561 and the elutant is discarded as waste. DNAextraction may be completed using washes from first column wash 565,second column wash 569, and water 571. Air from pneumatic interface 511can be forced through DNA extraction column 561 to remove volatileorganic compounds. Water 571 can be used to rinse the purified DNA(optionally including the use of a de-binding buffer or a modulator ofstringency) into output vial 589.

Coordination of the on-cartridge steps can be supported by an operationsdevice, such as a bench-top electro-mechanical device. The timing ofpneumatic injections, the breaking of ampules, and the piercing ofreagent reservoirs can be coordinated manually, or by a computer programor mechanical system. Cartridge 501 can include any suitable materials,shape, or dimensions. For example, in some embodiments, fluids arehandled in macrofluidic environments up until entered into magneticconcentrator 545 and are handled according to microfluidic principlesthereafter. In general, microfluidic may refer to sub-microlitersvolumes.

FIG. 6 gives a perspective view of an exemplary cartridge according tocertain embodiments. Methods for manufacturing and operating fluidicsystems are known and discussed in Fredrickson and Zan, 2004,Macro-to-micro interfaces for microfluidic devices, Lab Chip4(6):526-33; U.S. Pat. No. 8,105,783; U.S. Pat. No. 7,785,869; U.S. Pat.No. 7,745,207; U.S. Pat. No. 7,553,647; U.S. Pub. 2009/0227005; U.S.Pub. 2008/0241000; and U.S. Pub. 2008/0003564, the contents of each ofwhich are incorporated by reference in their entirety for all purposes.

FIG. 7 gives a schematic diagram of elements and steps of the inventionas discussed above with respect to FIG. 5.

While discussed above as magnetic beads, substrate 219 for capture ofpathogens 201 may be the floor of a chamber such as, for example, trapchamber 531. In other embodiments, the capture surface is the ceiling.The captured pathogens 201 can there be washed to remove non-specificanalytes and unbound entities.

In certain embodiments, fluidic cartridge 501 is designed to bedisposable. In this instance, once pathogens 201 have been lysed and theeluate collected, cartridge 501 can be discarded. In this manner, eachcartridge is intended for a single use, and the potential forcross-contaminating pathogens due to repeated use is eliminated.

The cartridge can be prepared from any material in the art suitable forcontaining liquids and withstanding the rigors of sonication. In certainaspects, the entire cartridge is made of plastic or an acrylic plasticpolymer. In other aspects, the bottom or top cover of the cartridge canbe prepared from a film such as the biaxially-oriented polyethyleneterephthalate (BoPET) sold under the trademark MYLAR while the rest ofthe cartridge is made of plastic or an acrylic plastic polymer. Incertain embodiments, the BoPET film constitutes the aforementionedstructural interface in contact with the sonicator transducer.

In certain embodiments, the cartridge is connected to a fluidic deviceor system configured to flow liquids into and out of the cartridge. Thefluidic system can comprise a pump that delivers liquid, air, gas,beads, reagents, electricity or other signals, light, power or acombination thereof to the cartridge. The fluidic system can have afirst tubing line connected to the inlet of the input channel, forflowing liquid into the cartridge. The other end of the first tubingline is connected to the pump. A second tubing line for fluid leavingthe cartridge is connected to the outlet of the output channel. Theother end of the second tubing line may also be connected to the pump orto a container for collecting exiting fluid. The fluidic systemfacilitates the delivery of the target/magnetic particle complexes intothe chamber, as well as any washing of captured complexes. Thecollection of lysate is also facilitated by the fluidic system.

Devices of the invention may also include a detection module. Thedetection module is a component in which molecules, cells, or otherparticles are to be detected, identified, measured or interrogated onthe basis of at least one predetermined characteristic. The molecules,cells, or other particles can be examined one at a time, and thecharacteristic is detected or measured optically, for example, bytesting for the presence or amount of a label. In some aspects, thedetection module is in connection with one or more detectionapparatuses. The detection apparatuses can be optical or electricaldetectors or combinations thereof. Examples of suitable detectionapparatuses include optical waveguides, microscopes, diodes, lightstimulating devices (e.g., lasers), photomultiplier tubes, andprocessors (e.g., computers and software), and combinations thereof,which cooperate to detect a signal representative of a characteristic,marker, probe, label, or reporter, and to determine and direct ameasurement or sorting action. However, other detection techniques canbe used as well.

Devices of the invention may include a computer component. For example,a user interface may be provided with input/output mechanisms (monitor,keyboard, touchscreen, etc.) coupled to a memory and a processor (e.g.,a silicone chip and a solid-state or magnetic hard drive). Input/outputmechanisms may be included for data, such as a USB port, Ethernet port,or Wi-Fi card or a hardware connection to a detection module, fluidicchip and pneumatic interface, or both.

In certain aspects, the detection module is in fluid connection with thefluidic cartridge. For example, the outlet of the fluidic cartridge maybe connected to the detection module by means of a tubing line. In thismanner, lysate leaving the cartridge can enter the detection modulewherein the contents of the lysate can be detected and analyzed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

1-13. (canceled)
 14. A method of isolating a microorganism in a sample,the method comprising: obtaining a sample suspected of containing two ormore microorganisms; introducing to the sample two or more viruses, eachvirus capable of causing a different microorganism to present a surfaceelement; exposing the sample to one or more capture moieties that bindthe surface elements presented on the microorganisms, thereby formingcapture moiety/microorganism complexes; and isolating the capturemoiety/microorganism complexes from the sample.
 15. The method of claim14, wherein the two or more viruses cause the two or more microorganismsto display the same element.
 16. The method of claim 14, wherein the twoor more viruses cause the two or more microorganisms to each display adifferent element.
 17. The method of claim 14, wherein at least one ofsaid two or more viruses is a bacteriophage.
 18. The method of claim 15,wherein the element is a cell surface protein.
 19. The method of claim16, wherein the different elements comprise different cell surfaceproteins.
 20. The method of claim 14, wherein the one or more capturemoieties comprise one or more antibodies.
 21. The method of claim 17,wherein said bacteriophage contains a biotinylation domain.
 22. Themethod of claim 14, wherein the at least one of the one or more capturemoieties comprises streptavidin.
 23. The method of claim 14, wherein atleast one of the two or more microorganisms is a bacteria.
 24. Themethod of claim 14, wherein at least one of the two or moremicroorganisms is a fungus.
 25. The method of claim 14, wherein the twoor more microorganisms are present in the sample at as low as 1 CFU/mL.26. The method of claim 14, wherein the one or more capture moieties arebound to magnetic particles.
 27. The method of claim 26, wherein saidisolating step comprises applying a magnetic field.